Towards Clinical Progress in the Mucopolysaccharidoses

Overview

The National Institute of Neurological Disorders and Stroke (NINDS), the Office of Rare Diseases (ORD), and the National Institute
of Diabetes and Digestive and Kidney Diseases at NIH along with the National MPS Society co-sponsored this workshop. This
scientific workshop was organized by Mark Haskins, V.M.D., Ph.D. (University of Pennsylvania); Sissi Langford (National MPS
Society); Catherine McKeon, Ph.D. (National Institute of Diabetes and Digestive and Kidney Diseases); Mark Sands, Ph.D. (Washington
University School of Medicine); and Danilo A. Tagle, Ph.D. (National Institute of Neurological Disorders and Stroke)

The goals of the workshop include:

The goal of this workshop is to identify and address impediments to effective therapies in the mucopolysaccharidoses (MPS)

To develop a strategy to determine the minimum standard protocol for clinical application of effective therapies for MPS and
other lysosomal storage disorders (LSDs).

Executive Summary

There was considerable progress reported at this workshop on both the clinical and basic research areas in the mucopolysaccharidoses
(see agenda). The Panel Discussions also identified areas of research that are required for efficient translation of new therapies
and approaches into the clinic.

Progress in basic and translational research:

A) Enzyme Replacement:William Sly presented preliminary data showing that completely de-glycoslyated -glucuronidase had a dramatically increased
half-life in serum and virtually eliminated lysosomal storage in the CNS of adult MPS VII mice. This surprising finding has
obvious and important implications for the treatment of the CNS disease associated with MPS disorders.

Elizabeth Neufeld presented the principles behind the development of aptamer-based modifications of recombinant α-L-iduronidase for treatment of the CNS disease associated with MPS I. Preliminary data generated in an in vitro blood brain barrier model shows that this approach may be able to deliver enzyme across the blood-brain barrier (BBB).

Patricia Dickson showed promising data using intrathecal delivery of α-L-iduronidase in the canine model of MPS I. The levels of glycosaminoglycans in the brain were normalized and the enzyme
persisted for 4-7 days.

B) Small Molecule Therapy:Thomas Seyfried showed that treatment with NB-DGJ, which interferes with the synthesis of gangliosides, decreased the levels
of gangliosides in the mouse models of GM1 gangliosidosis and Sandhoff disease. In addition, he showed that caloric restriction
decreased inflammation, improved rotarod performance, and increased the life span of these mice, however, there was no reduction
in gangliosides. Experiments are ongoing to determine the efficacy of combining substrate reduction and dietary restriction.

Steven Walkley showed that substrate reduction and by-product replacement therapies were partially effective in Niemann-Pick
type C disease. He also showed that combining the two approaches was more effective than either therapy alone.

David Bedwell developed a murine model that contained an authentic human non-sense mutation leading to MPS I. Using this model
he was able to test a safer (less toxic) analog of gentamycin for stop codon read-through. This safer drug was able to restore
approximately 3% of the enzyme activity in vitro and reduced urinary glycoaminoglycans (GAGs) in vivo.

C) Stem CellsKyuson Yun showed that fetal liver cells cultured with Noggin resulted in a 5- fold increase in the generation of neuronal
stem cells. Small numbers of these cells were seen in the brain following intracranial injection of transduced cells. Data
was also presented describing an easily accessible source (nasal epithelium) of autologous neuronal stem cells that express
the correct neuronal markers in vitro.

Raquel Walton presented in vitro data suggesting that there is a block in the maturation of neuronal stem cells derived from
several different areas of the MPS VII dog brain. When transplanted into the brains of MPS VII dogs, only GUSB-positive astrocytes
were observed and no GUSB-positive neurons were detected.

Nobuko Uchida presented pre-clinical data showing that human neuronal stem cells distributed widely throughout the neuraxis
and reduced the clinical signs associated with the Shiverer mouse. The cells also distributed similarly throughout the brains
in the mouse model of Infantile Batten Disease.

D) Gene Therapy:Miguel Sena-Esteves showed that direct intracranial injection of either AAV1 or AAV8 reduced ganglioside and cholesterol accumulation
in the brains of GM1 gangliosidosis mice. Retrograde transport of -galactosidase was observed with both vectors, and enzyme
activity was observed as distant as the retina and spinal cord following AAV8 injection.

Gordon Watson showed that intrathecal injection of an AAV vector resulted in persistent expression and widespread reduction
of lysosomal storage throughout the CNS in both the MPS VII and MPS I mouse models.

John Wolfe showed that intracranial injection of an AAV1 vector into the brain of -mannosidosis cats resulted in widespread
correction of disease, and dramatic clinical improvement of the animals. He also showed that a 1 l injection of AAV in the
ventral tegmental area, which sends projections to many areas of the brain, can deliver enzyme and reduce lysosomal storage
throughout the brain in MPS VII mice.

Kathy Ponder extended her studies using retroviral-mediated gene therapy in newborn MPS I dogs and showed that in addition
to correction in visceral organs, other tissues that have been refractory to conventional therapies (aorta and brain) also
respond to high expression during the newborn period. Alberto Auricchio, using MPS VI rats and cats, showed that AAV1 and
AAV8 expressing arylsulfatase B transduced the muscle and liver, respectively. He showed that each target tissue expressed
high levels of enzyme but arylsulfatase B was not secreted from muscle. Data describing the clinical response is still pending.

Jay Dritz showed pre-clinical data comparing the levels of expression of GUSB and GUSB-TAT in the hematopoietic compartment
following lentiviral mediated gene therapy. High level expression in multiple tissues was observed. Effects on critical tissues
such as the brain and kidney are pending. Jean-Michel Heard showed that there was persistent and widespread expression of
α-L-iduronidase in the brains of MPS I dogs following 8 separate injections of an AAV5 vector. Lysosomal storage was reduced
in targeted areas of the brain. However, immunosuppression (cyclosporine) was required to maintain expression.

Matthew Ellinwood presented similar data in the canine model of MPS IIIB. Three affected but pre-symptomatic dogs were injected
intracranially with an AAV5 vector and were maintained on an immunosuppressive regimen. There was widespread enzyme distribution
and their neurological exam was normal at sacrifice, however, significant inflammatory lesions were seen in the absence of
immunosuppression.

Xiucui Ma presented data showing that long term expression of α-L-iduronidase was possible in dogs with MPS I treated as young adults following transient treatment with anti-CD4 or anti-CD40
ligand combined with CTLA-4. Interestingly, data was also presented that showed improvements in skeletal disease, auditory-evoked
brain stem responses, and decreased storage in the brain. Adarsh Reddy showed that bone marrow transplantation or CNS-directed
AAV-mediated gene therapy alone in the murine model of globoid-cell leukodystrophy resulted in significant increases in life
span to an average of 45 and 55 days of age, respectively. However, when combined the two disparate therapies synergized dramatically
and resulted in an average life span of 105 days.

Mark Sands presented follow- up data on the original observation of hepatocellular carcinoma following systemic AAV-mediated
gene therapy. They repeated the original observation in a larger cohort of animals and showed that all of the AAV proviral
sequences integrated in a region of mouse chromosome 12 that contains a large number of microRNAs. These integration events
disrupted the normal expression pattern in that region.

E) Animal Models:Andrea Ballabio discussed the characterization of the multiple sulfatase deficiency mice. This mouse has a very severe phenotype
which includes systemic inflammation, neurodegeneration, and a defect in autophagy.

Progress in clinical research:

A) Enzyme Replacement:Three recombinant protein products are currently approved for clinical use for enzyme replacement therapy in MPS I (Aldurazyme),
MPS II (Elaprase) and MPS VI (Naglazyme). In general, the clinical response following intravenous infusion has been positive
with reduced hepatosplenomegaly, increased range of motion, increased stamina, and decreased urinary GAGs. Enzyme replacement
products are in development for MPS IV and MPS VII.

Patricia Dickson discussed the preliminary findings of a human clinical trial to test the safety of intrathecal delivery of
α-L-iduronidase. Three patients have been entered into the trial. So far the drug seems to be well tolerated with no severe
adverse reactions and two patients have shown some symptomatic improvement.

B) Gene Therapy:Ron Crystal discussed the preliminary data from an AAV2-mediated CNS-directed gene therapy clinical trial in children with
Late Infantile Neuronal Ceroid Lipofuscinosis. So far, the procedure has been well tolerated and there is preliminary evidence
that the procedure may be providing some efficacy. He also outlined improvements to the current protocol, specifically, the
use of a new AAV serotype (rh from rhesus) that results in higher level expression and distribution in the brain. Jean-Michel
Heard outlined an AAV5-mediated CNS-directed human gene therapy clinical trial for MPS I. This trial is based on the pre-clinical
data generated in the canine model of MPS I.

C) Stem Cell-Mediated Therapy:Nobuko Uchida presented preliminary data regarding a human clinical trial for Infantile Neuronal Ceroid Lipofuscinosis using
neuronal stem cells. Several children have been injected and there have been no severe adverse reactions as of yet. Clinical
evaluation is pending.

Joanne Kurtzberg discussed the use of umbilical cord blood for the treatment of lysosomal disorders. They have clear evidence
that early treatment (as early as several weeks or months of age) is critical for effective therapy. They have also initiated
a trial where they have added accessory cells that enhance early engraftment (data are pending).

D) Newborn Screening and developing Outcome MeasuresRonald Scott and John Hopwood gave updates on the progress with comprehensive newborn screening for lysosomal storage diseases.
John Hopwood discussed his plans for screening 100,000 newborns in Australia with his mass spectrometry approach.

Joe Muenzer, Chet Whitley and Maria Escolar discussed the importance of meaningful clinical outcomes and natural history collection
for the evaluation of various clinical trials.

Research Directions and Anticipated Needs:

The most important issues that the workshop participants expressed consist of 1) the need for natural history studies, 2)
newborn screening programs, and 3) meaningful clinical outcome measures.

It is becoming clear that initiating therapy as early as possible is critical for the effective treatment of these diseases.
This highlights the need for newborn screening and the collection of natural history data to move the novel therapies to the
clinic.

There is growing evidence that combining various therapeutic modalities (small molecule, enzyme replacement, bone marrow transplantation,
and gene therapy) can be synergistic and dramatically increase efficacy.

Over the last 5-10 years, the discussions have shifted from "how to treat these diseases" to "how do we get the therapies
into the clinic". This is a very encouraging sign!

Agenda

Workshop Objectives: To identify and address impediments to effective therapies, and to develop a strategy to determine the
minimum standard protocol for clinical application of effective therapies for MPS and other LSDs

Session III: Current Therapeutic StrategiesChair: Katherine Ponder, M.D.Goal: Identify the limitations and need for improvements in the traditional therapeutic approaches for MPS(15 minutes per speaker)

General discussion (20 minutes): What are the practical hurdles in these approaches for moving into the clinic?

12:00 - 1:00 p.m.

Lunch

1:00 - 3:15 p.m.

Session IV: Pre-Clinical Research Using Large AnimalsChair: Mark Haskins, V.M.D., Ph.D.Goal: Determine lessons learned from the preclinical use of dogs, cats and other large animal models(15 minutes per speaker)

General discussion (15 minutes): Can the positive results obtained in murine models of MPS be recapitulated in larger animal
models? How can the work on these models potentially help in designing trials that will work in children?

What are the research and resource needs that will facilitate translational and clinical research addressing the CNS issues
for MPS?What therapeutic strategies should be emphasized?Is gene therapy the most promising therapeutic approach to cure MPS and other LSDs?What other therapies hold the most promise for treatment and delivery of therapies to the brain?What are the limitations of these therapies?

General discussion (45 minutes): What will it take to cure MPS in children? How can the current research potentially help
in designing trials that will work in children?

10:15 - 10:30 a.m.

Break

10:30 - 12:00 a.m.

Session VIII: Early Intervention and Meaningful Clinical OutcomesChair: Joseph Muenzer, M.D., Ph.D.Goal: Strategize on the most effective ways for designing and implementing treatment strategies for MPS and other LSDs.(12 minutes per speaker)

What do investigators need to know to go from animal models to human trials?What will clinical trials look like?How can clinicians start to gather the functional outcomes for developing surrogate markers, standard protocol and assessment
measures to apply a treatment?Which of the LSDs will most likely to be ready for gene therapy and why?Are there MPS's that will not benefit from gene therapy? Which type(s)?How will the therapy translate to other LSDs?How can the research community work to move in this direction?